Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: Primer sequences of genes of interest (GOI).

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques:

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: Antibodies utilized in western blot assays.

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Western Blot

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: Analysis of S100a4 expression in two different mouse models of pulmonary fibrosis. (A) RNA was isolated from uninfected and from MHV-68-infected IFN-γR −/− (ko) and C57BL/6 wild-type (wt) mice at days 14 and 45 after infection. S100a4 expression was analyzed by qRT-PCR, normalized to the expression of β-actin, and depicted as relative fold changes. Results are derived from three mice per group and shown as mean ± SD. (B) Lung homogenates from uninfected mice and MHV-68-infected mice at the indicated time points were subjected to western blot analysis for the S100a4 protein (three mice per group). Blots were either incubated with an anti-S100a4-antibody or an anti-GAPDH antibody as loading control. (C) S100a4 protein was measured in bronchoalveolar lavage (BAL) fluids from uninfected or MHV-68-infected IFN-γR −/− (ko) and C57BL/6 mice (wt) at days 20, 63, and 100 after infection. Each symbol represents a mouse. Results are derived from three mice per group and shown as mean ± SD. Unpaired t -test was performed for statistical analysis (* denotes p < 0.05; ** denotes p < 0.01; n.s. denotes non-significance). (D) Protein levels of S100a4 were measured in BAL fluid from PBS or bleomycin-treated C57BL/6 mice at 14 days after instillation. Each symbol represents a mouse. Results are derived from nine mice per group and shown as mean ± SD. Unpaired t -test was performed for statistical analysis (*** denotes p < 0.001).

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Expressing, Isolation, Infection, Quantitative RT-PCR, Derivative Assay, Western Blot, Incubation, Control

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: Co-localization of S100a4 with Mac3 + macrophages in tissue sections from fibrotic mice. (A) MHV-68-infected IFN-γR −/− mice at 45 days after infection. (B) Bleomycin-treated C57BL/6 mice at 14 days after instillation. Green fluorescence: Mac3 antigen; red fluorescence: S100a4 antigen; blue fluorescence: diamidino-2-phenylindole-stained nuclei. In the merged image, yellow fluorescence indicates co-localization of S100a4 antigen and alveolar macrophages. All photomicrographs were taken at 200× magnification. Scale bar 50 µm.

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Infection, Fluorescence, Staining

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: S100A4 is expressed by CD163+ macrophages in tissue sections from human lungs. Representative immunofluorescent stainings of paraffin sections from donor (left-hand panels) and idiopathic pulmonary fibrosis (IPF) tissue (right-hand panels) including higher magnification inserts in the bottom row. S100A4 is shown in red, the macrophage marker CD163 in green, and diamidino-2-phenylindole (DAPI) in blue. Scale bar 50 µm.

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: S100a4 promotes activation of lung fibroblasts. (A) Primary lung fibroblasts were treated with various concentrations of recombinant S100a4 (0–3 µg/ml) for 24 h, and expression of α-SMA was assessed by qRT-PCR. Results are mean ± SD of duplicate samples from one experiment. (B) Cells were treated with 2 µg/ml recombinant S100a4 or with recombinant S100a4 in the presence of a S100a4 neutralizing antibody for 24 and 48 h, respectively. Cells were harvested and analyzed for expression of α-SMA and collagen I by western blot. S100a4 neutralization eliminated activation of lung fibroblasts. Results are representative of three independent experiments with similar results.

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Activation Assay, Recombinant, Expressing, Quantitative RT-PCR, Western Blot, Neutralization

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: S100a4 accelerates lung fibroblast proliferation and migration. (A) Primary lung fibroblasts were treated with 2 µg/ml recombinant S100a4 or with recombinant S100a4 in the presence of a S100a4 neutralizing antibody or with antibody alone for 72 h. Cell proliferation was analyzed using the XTT kit. Results are representative of three independent experiments with similar results. Shown are mean ± SD of five replicates from one experiment. Unpaired t-test was performed for statistical analysis (**** denotes p < 0.0001). (B) Direct migration of primary lung fibroblasts in the presence of 2 µg/ml recombinant S100a4 or recombinant S100a4 in the presence of the S100a4 neutralizing antibody was analyzed by wound healing assay. Wound closure was determined 24 h after scratching. Representative phase-contrast pictures of the cells at 0 (immediately after the scratch) and 24 h after the scratch are shown. The assay was performed three times: one representative experiment is presented. (C) For quantification, the wound area was measured using ImageJ and normalized to control at 0 h. Results are representative of three independent experiments with similar results. Shown are mean ± SD of triplicate samples from one experiment. The effect of stimulation by S100a4 was statistically significant in comparison to the control sample, as evaluated by the unpaired t -test (** denotes p < 0.01; *** denotes p < 0.001).

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Migration, Recombinant, Wound Healing Assay, Control, Comparison

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: Effect of conditioned medium on lung fibroblast proliferation. Primary lung fibroblasts were treated with conditioned medium (C.M.) from M0, M2, and M2 macrophages transfected with scrambled or S100a4-specific siRNA. In addition, specific S100a4 antibody or isotype control rabbit serum pre-treated M2 conditioned medium were used to stimulate lung fibroblasts. Cell proliferation was analyzed by using XTT kit after 48 h of treatments. Results are representative of two independent experiments with similar results. Shown are mean ± SD of five replicates from one experiment. Unpaired t -test was performed for statistical analysis (** denotes p < 0.01; ****denotes p < 0.0001).

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: Transfection, Control

Journal: Frontiers in Immunology

Article Title: S100a4 Is Secreted by Alternatively Activated Alveolar Macrophages and Promotes Activation of Lung Fibroblasts in Pulmonary Fibrosis

doi: 10.3389/fimmu.2018.01216

Figure Lengend Snippet: Niclosamide treatment in vivo. (A) Treatment with niclosamide improves survival of bleomycin-instilled mice. Mice were intratracheally treated with bleomycin, or as a control, with PBS. Beginning at day 7 after bleomycin instillation, mice were treated once daily with niclosamide (20 mg/kg) or vehicle. Mice were monitored daily for signs of disease, and any mice that appeared moribund were sacrificed. Treatment with niclosamide significantly improved the survival of bleomycin-instilled mice [* p = 0.031; Log-rank (Mantel–Cox) test]. Numbers in brackets indicate the total number of mice per group. (B,C) Treatment with niclosamide reduces S100a4 protein in the BAL fluid. The surviving mice depicted in panel (A) were subjected to BAL. The protein concentration of S100a4 in the BAL fluids was determined by ELISA. Each symbol represents a mouse. Results are shown as mean ± SD. Unpaired t -test was performed for statistical analysis (*** p < 0.001; **** p < 0.0001). (D) Treatment with niclosamide improves lung function. The surviving mice depicted in panel (A) were subjected to a lung function test, and lung compliance was determined. Each symbol represents a mouse. Results are shown as mean ± SD. Unpaired t -test was performed for statistical analysis (* p < 0.05; ** p < 0.01).

Article Snippet: Briefly, primary lung fibroblast cells were plated into 96-well plates (3 × 10 3 cells/well) in complete DMEM/F-12 medium (Gibco) and allowed to accommodate overnight before quiescing by replacing with serum-free medium for a further 12 h. Subsequently, cells were incubated in 2% FBS DMEM/F-12 medium with or without recombinant S100a4 protein (2 μg/ml) or together with the anti-S100a4 antibody (3 μg/ml) (R&D Systems) for 72 h. Prior to harvesting, cells were treated with XTT labeling mixture for 4 h, and the absorbance was quantified at 450 nm with a reference wave length at 650 nm by using a microplate absorbance reader (TECAN SUNRISE).

Techniques: In Vivo, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Journal of pharmacological sciences

Article Title: Lymphangiogenesis induced by vascular endothelial growth factor receptor 1 signaling contributes to the progression of endometriosis in mice.

doi: 10.1016/j.jphs.2020.05.003

Figure Lengend Snippet: Fig. 3. Accumulation of macrophages and fibroblasts in endometrial implant tissues. (A) The levels of the mRNAs encoding the lymphangiogenic growth factors VEGF-C and VEGF-D in endometrial tissues from WT/WT and TK-/-/TK-/- mice. Data are expressed as the mean ± SEM (n ¼ 5e6 mice per group). *P < 0.05. (B) Double immunostaining of VEGF-C (green) or VEGF-D (green), and CD11b (red) or S100A4 (red) in endometrial tissues from WT/WT mice at Day 14. Scale bars, 50 mm. (C) Left: Immunostaining of CD11b (green) in endometrial tissues from WT/WT and TK-/-/TK-/- mice at Day 14. Scale bars, 100 mm. Right: The numbers of CD11bþ cells in endometrial tissues from WT/WT and TK-/-/TK-/-

Article Snippet: The sections were incubated with the following primary antibodies at 4 C overnight: a rabbit anti-mouse VEGFR1 polyclonal antibody (1:200; Abcam), a goat anti-mouse VEGFR1 polyclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit anti-LYVE-1 polyclonal antibody (1:200; Abcam), a goat anti-LYVE-1 polyclonal antibody (1:200; R&D Systems Inc., Minneapolis, MN, USA), a rabbit anti-VEGF-C polyclonal antibody (1:200; Abcam), a goat anti-VEGFD polyclonal antibody (1:40; R&D Systems Inc.), a rat anti-mouse CD11b monoclonal antibody (1:200; BD Biosciences, San Jose, CA, USA), a rabbit anti-mouse S100A4 polyclonal antibody (1:200; Abcam), or a goat anti-mouse S100A4 polyclonal antibody (1:100; OriGene Technologies, Rockville, MD, USA).

Techniques: Double Immunostaining, Immunostaining

Journal: Journal of Radiation Research

Article Title: Chk1 suppression leads to a reduction in the enhanced radiation-induced invasive capability on breast cancer cells

doi: 10.1093/jrr/rrab049

Figure Lengend Snippet: Combined effects of PD407824 and γ-irradiation on the expression and activities of S100A4 and MMP-2 on MDA-MB-231 cells. Non-IR = no irradiation, PD = PD407824. * P < 0.05 vs Non-IR † P < 0.01; error bar means standard deviation. All experiments conducted three times independently. (A) Effects of PD407824 on S100A4 and MMP-2 mRNA levels. (B) Effects of PD407824 combined with γ-irradiation on S100A4 and phosphorylation of Chk1. (C) Effect of PD407824 on MMP-2 activity. (D) Effects of a Chk1-specific siRNA (siChk1) on S100A4 and p-Chk1 expression. (E) Effect of siChk1 on MMP-2 activity.

Article Snippet: Primary antibodies were against S100A4 (Sheep IgG, Cat. # AF4138, R&D Systems, Minneapolis, MN, USA), p-Chk1 (Ser345) (Rabbit IgG, Cat. # 2348, Cell Signaling, Danvers, MA, USA), Chk-1 (Mouse IgG, Cat. # 2360, Cell Signaling) (dilute to 1:1000), and β-actin (Mouse IgG, Cat. # ab8226, Abcam, Cambridge, UK) (dilute to 1:10000).

Techniques: Irradiation, Expressing, Standard Deviation, Phospho-proteomics, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury

doi: 10.1038/s41418-019-0336-3

Figure Lengend Snippet: Both mTORC1 and mTORC2 signaling are activated in kidney fibroblasts after IRI. a The graph showing the blood urea nitrogen (BUN) level in CD-1 mice at day 1 after UNx or IRI. *P < 0.05, n = 3 (left). Kidney histology as shown by periodic acid-Schiff (PAS) staining. Scale bar = 20 µm (right). b, c Western blot analyses showing the induction of p-Akt (Ser473), and p-S6 in the kidneys after ischemia-reperfusion injury (IRI). The numbers indicate each individual animal within the given group (b). The samples were pooled from three animals within each group (c). d Representative images showing the induction for p-Akt (Ser473) and p-S6 in Gli1- or Fsp1-positive fibroblasts from the IRI kidneys. White arrows indicate the co-staining positive cells. Scale bar = 20 µm

Article Snippet: After blocking with 2% normal donkey serum for 60 min, the slides were immunostained with antibodies against Gli1 (cat: 388516, RD Systems), Fsp1 (cat: NB100-55404, Novus), PDGFRβ (cat:14-1402, eBioscience), p-Akt (Ser473) (cat: 3868, Cell Signaling Technology), p-S6 (cat: 4858, Cell Signaling Technology), Rheb (cat: ab25873, abcam), Tsc1 (cat: 4906, Cell Signaling Technology), Rictor (cat: A300-459A, Bethyl Laboratories), cleaved caspase 3 (cat: 9664, Cell Signaling Technology), Ly6b (cat: MCA771G, AbD Serotec, Raleigh, NC), and CD3 (cat: 555273, BD Pharmingen), respectively.

Techniques: Staining, Western Blot